Nematodes are a very large group of invertebrate animals generally referred to as roundworms, threadworms, eelworms or nematodes. Some nematodes are plant parasites and can feed on stems, buds, leaves, and in particular on roots. One important genus of plant parasitic nematodes is the root-knot nematode (Meloidogyne spp). These parasitic nematodes infect a wide range of important field, vegetable, fruit and ornamental plants.
Various methods based on chemicals, pesticides or fumigants are present for treating or preventing root-knot nematode disease in plants. Chemical agents are often not selective, and exert their effects on non-target organisms, effectively disrupting populations of beneficial microorganisms, for a period of time following application of the agent. Chemical agents may persist in the environment and only be slowly metabolized. The fumigant agent such as chloropicrin is effective in controlling root-knot and other plant-parasitic nematodes. However, this approach is costly and also difficult to apply properly under the prevailing conditions. U.S. Pat. No. 5,013,762 discloses a chemical based method using Bromonitromethane as the active agent for the treatment of nematodes.
The biological control organisms have also been used to try to control nematode disease in crops. Commercially available preparations of biological control organisms are limited in their use to regions that can support the growth of the control organism. Moreover, the outcome of using one organism to control another is unpredictable and subject to a variety of factors such as weather and climate.
Additionally, among plant parasitic nematodes, the RKNs are a leading cause of crop losses. There are several examples of host genes conferring resistance to nematodes in diverse crops. The availability of host plant resistance is substantially limited with appropriate resistance loci lacking for the majority of crops (Roberts, P. A. 1992. Journal of Nematology 24:213-227). In addition, the resistance is limited to only a few RKN species or populations and some resistance genes are heat-sensitive and thus unsuitable for hot production areas. Another limitation of natural resistance genes is the durability of resistance since resistance-breaking populations of RKN can develop after continuous exposure to resistant cultivars, e.g. root-knot resistant tomatoes. Furthermore, there are six stages and four molts in the development of root knot nematode i.e. egg stage; J1 (i.e. first juvenile stage); M1 (i.e. first molt); J2 (second juvenile stage; sometimes hatch from egg); M2; J3; M3; J4; M4; A (adult). The gene expression may be specific to one or more lifecycle stages only.
FMRFamide (Phe-Met-Arg-Phe) is a neuropeptide from a broad family of FMRFamide-related peptides (FaRPs) all sharing an -RFamide sequence at their C-terminus. FMRFamide like peptides (FLPs) belong to FaRPs comprising the largest family of neuropeptides in nematodes. Most of the structural information of FLPs has been generated from Caenorhabditis elegans and the functional data of FLPs comes from the nematode physiological model, Ascaris suum. The studies on FLPs in A. suum indicate that their vital responsibility for the modulation of nerve and muscle activity in a concentration dependent and reversible manner [Day T. A and Maule A. G (1999), Peptides Vo. 20, pages 999-1019) and Geary T. G et al. (1999), Ann. N Y Acad. Sci. Vol. 897, pages 212-227)]. They are responsible for modulating pharyngeal muscle activity in A. suum [Brownlee D. J et al. (1996), Parasitol. Today, vol. 12, pages 343-351]. Similarly, FLPs are also accountable for multiple responses in the ovijector musculature ranging from transient excitation to persistent or transient inhibition [Fellowes R. A et al. (1998) Parasitology 116 (Pt3): 277-2878; Fellowes R. A et al. (2000). Parasitology 121 (Pt 3): 325-336.]. Therefore, these peptides and their associated processes are considered as potential control targets for parasitic helminths [McVeigh P et al. (2006). Trends Parasitol 22: 385-396; Maule A. G. et al. (2002) Curr Top Med Chem 2: 733-758; Mousley A, et al. (2005) Int J Parasitol 35:1557-1567.]. As in the case of A. suum, presence of FMRFamide-like immunoreactivity has also been demonstrated in the nervous system of PPNs, Globodera pallida and G. rostochiensis [Kimber M. J. et al. (2001). Mol Biochem Parasitol 116: 199-208.]. Recently it has been reported that flp-32 in G. pallida was responsible for the modulation of locomotory behavior and putatively interacted with at least one novel G-protein coupled receptor [Xue B. et al (2013). Phytopathology 103: 175-181].
FLPs are shown to be present in different parasitic nematodes having similar structural homologues and functions. Consequently, disruption of these activities in PPNs represents an attractive novel control strategy as it would interrupt the worm's ability to hatch, migrate through the soil to reach the host, feed on the host tissue and also to mate. So far, 19 FLPs have been identified in M. incognita based on conserved FMRFamide domain analysis of the ESTs and the whole genome sequence by comparative genomics [Abad P et al. (2008). Nat Biotechnol 26: 909-915], out of which six have transcriptional confirmation (flp-1, 7, 12, 14, 16, 18—NCBI GenBank database). MSA (Multiple Sequence alignment) of these six confirmed flp genes showed low nucleotide sequence level conservation among them although they share a common RF-amide sequence at C-terminus. Their uniqueness could therefore be harvested at developing sequence specific knockout module by dsRNA method to avoid off target effects. Further, accurate physiological roles of only few of the FLPs are known and the major information is lacking in M. incognita probably due to their small size and obligate relationship with the host which limits the use of standard physiological techniques. Nevertheless, silencing of flp-14 and flp-18 in M. incognita has been reported to interrupt the migration of worms in response to the root exudates [Dalzell J J et al. (2010). Int J Parasitol 40: 91-100.; Dalzell J J et al. (2009). Int J Parasitol 39: 1503-1516.].
Charlton et al. (2010) showed that suppression of two M. incognita genes (dual oxidase and a subunit of a signal peptidase required for the processing of nematode secreted proteins) using RNAi resulted in the reduction in the number of nematodes by 50%.
U.S. Pat. No. 6,506,559 demonstrates the effectiveness of RNAi against known genes in C. elegans. However, it does not reveal any kind of usefulness of RNAi for controlling plant parasitic nematodes.
WIPO patent application WO04/005485 also discloses RNA interference (RNAi) based process for the reduction in nematode reproduction. But, the process does not provide complete effectiveness in reducing nematodes.
US20100068172 relates to methods for controlling nematode infestation via dsRNA mediated gene silencing; whereby nematodes were incubated in the double stranded RNA and whereby the double-stranded RNA is taken up by the nematodes. In one particular embodiment; the methods of the invention are used to alleviate plants from nematode pests. Alternatively; the methods are used for treating and/or preventing nematode infestation on a substrate or a subject in need of such treatment and/or prevention. Suitable nematode target genes and fragments thereof; dsRNA constructs; recombinant constructs and compositions are disclosed.
US20120030834 relates to achieving a plant protective effect through the identification of target coding sequences and the use of recombinant DNA technologies for post-transcriptionally repressing or inhibiting expression of the target coding sequences in the cells of plant-parasitic nematodes. The disclosed gene targets show significant conservation at the nucleotide level between orthologs from different Meloidogyne species, facilitating genus-wide targeting by RNA interference.
US20080184391 relates to a transgenic plant having a nucleic acid molecule of a pathogen, wherein the transgenic plant has increased resistance to infection by the pathogen. The nucleic acid molecule of the pathogen is one of (a) a portion of a polynucleotide coding for a protein of the pathogen; (b) a complementary sequence to (a); (c) a combination of (a) and (b); and (d) a combination of (a) and (b) further having a spacer nucleotide sequence. The protein of the pathogen is one of a protein involved in chromatin remodeling in the pathogen, a protein in a recombination pathway of the pathogen, a protein in a nucleotide repair pathway of the pathogen, a protein for post transcriptional processing of RNA in the pathogen, or combinations thereof.
EP1799029 relates to compositions for controlling plant parasites and compositions for increasing root growth, more particularly to nucleic acid compositions for controlling nematode disease or increasing root growth.
US20110150839 relates to a method to obtain transgenic plants resistant to the attack of phytopathogens (i.e. parasites and phytophages) based on RNA interference, Which contemplates the expression of double strand RNA (dsRNA) in the plant tissues, suitable for inhibiting the functionality of a GPCR receptor, Whose functioning is vital for fungi, herbivorous insects or phytopathogenic nematodes.
US20030150017 relates to methods which provide for the genetic control of pathogen infestation in host organisms such as plants, vertebrate animals and fungi. Such methods utilize the host as a delivery system for the delivery of genetic agents, preferably in the form of RNA molecules, to a pathogen, which agents cause directly or indirectly impairment in the ability of the pathogen to maintain itself, grow or otherwise infest a host plant, vertebrate animal or fungus. Also provided are DNA constructs and novel nematode nucleotide sequences for use in same, which facilitate pathogen resistance when expressed in a genetically-modified host. Such constructs direct the expression of RNA molecules substantially homologous and/or complementary to an RNA molecule encoded by a nucleotide sequence within the genome of a pathogen and/or of the cells of a host to effect down regulation of the nucleotide sequence. Particular hosts contemplated are plants, such as pineapple plants, and particular pathogens are nematodes.
CN102352357 relates to a method for inhibiting gene dsRNA of neuropeptide FLPs its application. The invention provides larvae of M. incognita in solution of dsRNA reduces root nematodes tendency to migrate host plants, to reduce host plants colonization, and also inhibit nematode reproduction.
Thus, there is a need of an effective method for the inhibition of RKNs, which is capable of achieving the reduction in the multiplication of nematodes at multiple stages and can ultimately help in the reduction of multiple factors/components like the simultaneous decrease in the female population laying eggs, decrease in egg production etc. so as to ensure the complete and successful elimination of the infecting nematodes.